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<h4 class="current" id="try">Biofilm</h4>

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<h1>Design</h1>
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<h1>Bubbling Method</h1>
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<p><img
	src="http://2011.igem.org/wiki/images/9/92/Zju-biofilm03.jpg"
	style="width: 350px; float: right"/>First we inoculated 1%
overnight cultured E.coli DH5α in 60ml LB (depends on the volume of tube
used). Then we placed a glass slide inside the tube, leaving a short end
outside the liquid. With a common air pump for aquarium, an ultrafilter
membrane and rubber tubes we constructed a bubble supply system. And air
bubbles were pumped into the culture medium. We placed the set in a
clean bench to prevent contamination since the tube was not strictly
isolated. The formation can take place at about 27℃. Biofilm was
generally formed in 72 hours. It was usually small clusters of bacteria
clinging to the glass slide. We used a blank slide, placed it in the
culture and compared with the other one to see whether the bacteria
simply clung to the glass slide or they had formed biofilm on it. </p>
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<h1>Rubber Tube Method</h1>
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<p><img
	src="http://2011.igem.org/wiki/images/1/11/Zju-biofilm01.jpg"
	style="width: 350px; float: right" />We used a glass ware with two
opening at the bottom to attach rubber tube and used rubber tube of
2mm*4mm(inside diameter*outside diameter) and 1mm*2mm to make a
circulating flowing incubator. We then sealed all connecting locations
with parafilm. The set was inoculated with 1% overnight cultured DH5ɑ in
and sealed with parafilm. We then placed it in 37℃ for 3 hours for
E.coli to attach. We used a mounted pump to create a liquid flow of
culture medium. The flowing speed should be about 60ml per 24 hours.
Biofilm was formed in about 48 hours and white substance was visible in
the inner tube. We cut them with scissors from the long tube at two ends
at the same time and cut open along the long axis. This prevented the
sudden flow of water from destroying the structure of biofilm. Then we
sliced the tube with frozen tissue slicer to into pieces of 50μm and
observed under regular microscopy for vertical structure.</p>
<p></p>
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<h1>Cell Culture Plate Method</h1>
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<p><strong>Microbial inoculation</strong><br />
<img src="http://2011.igem.org/wiki/images/8/85/Zju-biofilm04.jpg"
	style="width: 350px; float: right;" /> For every experiment, the cells
(modified Escherichia.coli, DH5alpha) were cultured in Lysogeny broth
(LB) medium overnight (14-18 hours) at 37 C, 200 rpm. The initial
culture was 100* diluted into new LB medium next morning and cultured in
the same conditions until the cells grew to OD600 0.5-0.7.</p>
<p><strong>Biofilm formation</strong><br />
20 L of the cell culture of OD600 0.5-0.7 were then added into each well
of a 6-well plate or 24-well cell culture plate (Costar, Corning
Incorporated, USA) containing 2 mL LB medium and small pieces of glass
(cover slip), Polyethylene terephthalate (PETE) and PC (poly carbonate).
The cell culture plates were placed in the incubator at 37 C for 24
hours, 48 hours and 72 hours. Glass, PETE and PC coupons were taken out
from the culture afterwards w/ or w/o washing in distilled water.</p>
<p><strong>Confocal laser scanning microscopy</strong><br />
The CLSM images of biofilm were obtained with Zeiss LSM 710NLO confocal
laser scanning microscope using a 20⨉ water-immersion objective, or a
63X oil-immersion objective, or with Zeiss LSM510 Meta confocal laser
scanning microscope using a 63X oil-immersion objective. The
mono-florescence images, multichannel simulated fluorescence projection
images and vertical cross sections of the biofilm were obtained by
utilizing the software ZEN.</p>
<p><strong>Using AI-2 as biofilm formation accelerator</strong><br />
Quorum sensing plays an important role in biofilm formation. In many
Gram-negative bacteria, quorum-sensing systems respond to N-acyl
homoserine lactones (AHLs), which were proved to be essential for the
architecture of biofilm (Dickschat JS, 2010; Lopez D, Vlamakis H and
Kolter R, 2010). In addition, there has been study indicated the
universal quorum sensing signaling molecule autoinducer 2 (AI-2) can
increase E.Coli’s biofilm biomass 30-folds (Gonzalez Barrios AF, 2006).
Thus we co-transformed our device along with BBa_K091191, a device
capable of sending out AI-2 molecules under the control of a pBAD
promoter that can be induced with arabinose. We also did
co-transformation with our device and another device from the registry
consisting of LuxI under the control of a lacI repressible promoter.</p>
<p></p>
<div id="importantcontainer">
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<h1>How to set up a biofilm reactor in your lab: Shopping list</h1>
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<p id="framet1">Bubbling method</p>
<table width="0" border="1" cellspacing="1" cellpadding="1">
	<tr>
		<td>
		<p>Large test tubes with plug</p>
		<a href="http://item.taobao.com/item.htm?id=12269690038">http://item.taobao.com/item.htm?id=12269690038</a></td>
		<td>
		<p>$0.43</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Rubber tube</p>
		<a href="http://item.taobao.com/item.htm?id=12473511203">http://item.taobao.com/item.htm?id=12473511203</a></td>
		<td>
		<p>$0.32/meter (mailing cost not included) 0.4 meter per usage</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Glass slide</p>
		<a href="http://item.taobao.com/item.htm?id=8877150757">http://item.taobao.com/item.htm?id=8877150757</a></td>
		<td>
		<p>$0.94 for 50 slides</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Common lab ware: (Test tubes, a shaker, an incubator, pipettes
		and culturing medium)</p>
		</td>
		<td>We believe you already have them</td>
	</tr>
	<tr>
		<td>
		<p>Total cost</p>
		</td>
		<td>
		<p>$0.58 per set</p>
		</td>
	</tr>
</table>
<p></p>
<p id="framet1">Rubber Tube method</p>
<table width="0" border="1" cellspacing="1" cellpadding="1">
	<tr>
		<td>
		<p>Silicon tube</p>
		<a href="http://item.taobao.com/item.htm?id=10401921234&">http://item.taobao.com/item.htm?id=10401921234&</a></td>
		<td>
		<p>$0.32/meter (mailing cost not included) 0.6 meter per usage</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Glassware</p>
		</td>
		<td>
		<p>Need to be made to order, prize may vary</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Wriggle pump</p>
		<a href="http://item.taobao.com/item.htm?id=1484983763">http://item.taobao.com/item.htm?id=1484983763</a></td>
		<td>
		<p>$217 (mailing cost not included) can be used on two sets of
		experiments at the same time</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Common lab ware: <br />
		(Test tubes, a shaker, an incubator, pipettes and culturing medium)</p>
		</td>
		<td>
		<p>We believe you already have them</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Total cost</p>
		</td>
		<td>
		<p>$218 per 2 sets</p>
		</td>
	</tr>
</table>
<p></p>
<p id="framet1">For Cell Culture Plate Method</p>

<table width="0" border="1" cellspacing="1" cellpadding="1">
	<tr>
		<td>
		<p>A 24 well, flat bottom Corning? Costar? cell culture plate;Or a
		6 well, flat bottom Corning? Costar? cell culture plates</p>
		</td>
		<td>
		<p>$3</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>10 cover slips</p>
		</td>
		<td>
		<p>$0.5</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Common lab ware: <br />
		(Test tubes, a shaker, an incubator, pipettes and culturing medium)</p>
		</td>
		<td>
		<p>We believe you already have them</p>
		</td>
	</tr>
	<tr>
		<td>
		<p>Total cost</p>
		</td>
		<td>
		<p>$3.5 per set!</p>
		</td>
	</tr>
</table>


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